ArticlePharmaceutics2022
Ribozyme Assays to Quantify the Capping Efficiency of In Vitro-Transcribed mRNA.
Article in Pharmaceutics, 2022. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 35 papers.
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Who cites it
35 citing papers in PubMed, 44 citations in OpenAlex.
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- A Modular Mechanistic In Silico Model for In Vitro Transcription Process Yield and Product Quality Prediction.Biotechnology and bioengineering · 2026Article
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- Engineered T7 RNA polymerase to improve mRNA capping efficiency and reduce dsRNA generation duringMolecular therapy. Advances · 2026Article
- Transcript-independent quantitative analysis of RNA cap structures by LC-MS/MS.Molecular therapy. Advances · 2026Article
- Nanopore direct RNA sequencing and the epitranscriptome: Advances in mapping native RNA landscapes.iMeta · 2026Review
- mRNA vaccine development for tuberculosis: antigen design, immune mechanisms, and translational challenges.Archives of microbiology · 2026Review
- Systematic screening of archaeal MazF homologs reveals Tth-MazF1, a versatile, sequence-specific ribonuclease from Thermococcus thioreducens.Nucleic acids research · 2026Article
- Process and analytical strategies for the safe production of mRNA vaccines and therapeutics.Molecular biology reports · 2026Review
- Novel trinucleotide mRNA capping reagents: improved synthetic route and efficient cotranscriptional incorporation in mRNA.RNA (New York, N.Y.) · 2026Article
- The mRNA-Based Innovative Strategy: Progress and Challenges.Nano-micro letters · 2026Review
- Engineering bone tissue with mRNA: from molecular design and delivery to clinical applications.Materials today (Kidlington, England) · 2025Article
- mRNA vaccines: immunogenicity and quality characteristics.Journal of nanobiotechnology · 2025Review
- Characterisation and analysis of mRNA critical quality attributes using liquid chromatography based methods.Journal of chromatography. A · 2025Review
- Optimal Chimeric Antigen Receptor (CAR)-mRNA for Transient CAR T Cell Generation.International journal of molecular sciences · 2025Article
- Generating mRNA Encoding Anti-HBV Designer Epigenome Modifiers.Methods in molecular biology (Clifton, N.J.) · 2025Article
- Translation of HIV-1 unspliced RNA is regulated by 5' untranslated region structure.Journal of virology · 2024Article
- Using nucleolytic toxins as restriction enzymes enables new RNA applications.Nucleic acids research · 2024Article
- From structural design to delivery: mRNA therapeutics for cancer immunotherapy.Exploration (Beijing, China) · 2024Review
Corrections and comments
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Authors and funding
11 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
The presence of the cap structure on the 5'-end of in vitro-transcribed (IVT) mRNA determines its translation and stability, underpinning its use in therapeutics. Both enzymatic and co-transcriptional capping may lead to incomplete positioning of the cap on newly synthesized RNA molecules. IVT mRNAs are rapidly emerging as novel biologics, including recent vaccines against COVID-19 and vaccine candidates against other infectious diseases, as well as for cancer immunotherapies and protein replacement therapies. Quality control methods necessary for the preclinical and clinical stages of development of these therapeutics are under ongoing development. Here, we described a method to assess the presence of the cap structure of IVT mRNAs. We designed a set of ribozyme assays to specifically cleave IVT mRNAs at a unique position and release 5'-end capped or uncapped cleavage products up to 30 nt long. We purified these products using silica-based columns and visualized/quantified them using denaturing polyacrylamide gel electrophoresis (PAGE) or liquid chromatography and mass spectrometry (LC-MS). Using this technology, we determined the capping efficiencies of IVT mRNAs with different features, which include: Different cap structures, diverse 5' untranslated regions, different nucleoside modifications, and diverse lengths. Taken together, the ribozyme cleavage assays we developed are fast and reliable for the analysis of capping efficiency for research and development purposes, as well as a general quality control for mRNA-based therapeutics.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.